Biotechnology for Biofuels and Bioproducts

Monogalactosyldiacylglycerol (MGDG), a predominant photosynthetic membrane lipid derived from plants and microalgae, has important applications in feed additives, medicine, and other fields. The low content and various structural stereoselectivity differences of MGDG in plants limited the biological extraction or chemical synthesis of MGDG, resulting in a supply shortage of monogalactosyldiacylglycerol with a growing demand. Herein, we established Saccharomyces cerevisiae as a cell factory for efficient de novo production of monogalactosyldiacylglycerol for the first time. Heterologous production of monogalactosyldiacylglycerol was achieved by overexpression of codon-optimized monogalactosyldiacylglycerol synthase gene MGD1, the key Kennedy pathway genes (i.e. GAT1, ICT1, and PAH1), and multi-copy integration of the MGD1 expression cassette. The final engineered strain (MG-8) was capable of producing monogalactosyldiacylglycerol with titers as high as 16.58 nmol/mg DCW in a shake flask and 103.2 nmol/mg DCW in a 5 L fed-batch fermenter, respectively. This is the first report of heterologous biosynthesis of monogalactosyldiacylglycerol in microorganisms, which will provide a favorable reference for study on heterologous production of monogalactosyldiacylglycerol in yeasts.

Xiaosong Gu

Yumei Shi

Changxin Luo

Jintao Cheng

Establishment of Saccharomyces cerevisiae as a cell factory for efficient de novo production of monogalactosyldiacylglycerol

建立酿酒酵母作为细胞工厂高效从头合成单半乳糖二酰甘油

Filamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications.A novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%.This PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications.

Lu Wang

Yijia Xie

Jingjing Chang

Juan Wang

Hong Liu

Mei Shi

Yaohua Zhong

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

一种新型蔗糖诱导表达系统和其在黑曲霉中生产生物质降解酶的应用

Yeast was the first microorganism used by mankind for biotransformation processes that laid the foundations of industrial biotechnology. In the last decade, Pichia pastoris has become the leading eukaryotic host organism for bioproduct generation. Most of the P. pastoris bioprocess operations has been relying on toxic methanol and glucose feed. In the actual bioeconomy era, for sustainable value-added bioproduct generation, non-conventional yeast P. pastoris bioprocess operations should be extended to low-cost and renewable substrates for large volume bio-based commodity productions. In this review, we evaluated the potential of P. pastoris for the establishment of circular bioeconomy due to its potential to generate industrially relevant bioproducts from renewable sources and waste streams in a cost-effective and environmentally friendly manner. Furthermore, we discussed challenges with the second generation P. pastoris platforms and propose novel insights for future perspectives. In this regard, potential of low cost substrate candidates, i.e., lignocellulosic biomass components, cereal by-products, sugar industry by-products molasses and sugarcane bagasse, high fructose syrup by-products, biodiesel industry by-product crude glycerol, kitchen waste and other agri-food industry by products were evaluated for P. pastoris cell growth promoting effects and recombinant protein production. Further metabolic pathway engineering of P. pastoris to construct renewable and low cost substrate utilization pathways was discussed. Although, second generation P. pastoris bioprocess operations for valorisation of wastes and by-products still in its infancy, rapidly emerging synthetic biology tools and metabolic engineering of P. pastoris will pave the way for more sustainable environment and bioeconomy. From environmental point of view, second generation bioprocess development is also important for waste recycling otherwise disposal of carbon-rich effluents creates environmental concerns. P. pastoris high tolerance to toxic contaminants found in lignocellulosic biomass hydrolysate and industrial waste effluent crude glycerol provides the yeast with advantages to extend its applications toward second generation P. pastoris strain design and bioprocess engineering, in the years to come.

Burcu Gündüz Ergün

Kübra Laçın

Buse Çaloğlu

Barış Binay

Second generation Pichia pastoris strain and bioprocess designs

第二代毕赤酵母菌株与生物工艺设计

O-Acetyl-L-homoserine (OAH) is an important potential platform chemical. However, low levels of production of OAH are greatly limiting its industrial application. Furthermore, as a common and safe amino acid-producing strain, Corynebacterium glutamicum has not yet achieved efficient production of OAH.First, exogenous L-homoserine acetyltransferase was introduced into an L-homoserine-producing strain, resulting in the accumulation of 0.98 g/L of OAH. Second, by comparing different acetyl-CoA biosynthesis pathways and adding several feedstocks (acetate, citrate, and pantothenate), the OAH titer increased 2.3-fold to 3.2 g/L. Then, the OAH titer further increased by 62.5% when the expression of L-homoserine dehydrogenase and L-homoserine acetyltransferase was strengthened via strong promoters. Finally, the engineered strain produced 17.4 g/L of OAH in 96 h with acetate as the supplementary feedstock in a 5-L bioreactor.This is the first report on the efficient production of OAH with C. glutamicum as the chassis, which would provide a good foundation for industrial production of OAH.

Ning Li

Weizhu Zeng

Jingwen Zhou

Sha Xu

O-Acetyl-L-homoserine production enhanced by pathway strengthening and acetate supplementation in Corynebacterium glutamicum

谷氨酸棒杆菌中通过途径强化和乙酸补充增强O-乙酰-L-高丝氨酸生产

Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation. Specifically, glucose oxidation to gluconic acid substantially lowers environmental pH and hinders BC production. Conversely, ethanol supplementation is known to enhance BC yields inKomagataeibacter spp.by promoting efficient glucose utilization.
K. sucrofermentansATCC 700178 was engineered, knocking out the four PQQ-mDHs, to assess their impact on BC production. The strain KS003, lacking PQQ-dependent glucose dehydrogenase (PQQ-GDH), did not produce gluconic acid and exhibited a 5.77-fold increase in BC production with glucose as the sole carbon source, and a 2.26-fold increase under optimal ethanol supplementation conditions. In contrast, the strain KS004, deficient in the PQQ-dependent alcohol dehydrogenase (PQQ-ADH), showed no significant change in BC yield in the single carbon source experiment but showed a restrained benefit from ethanol supplementation.
The results underscore the critical influence of PQQ-GDH and PQQ-ADH and clarify the effect of ethanol supplementation on BC production inK. sucrofermentansATCC 700178. This study provides a foundation for further metabolic pathway optimization, emphasizing the importance of diauxic ethanol metabolism for high BC production.

Pedro Montenegro-Silva

Tom Ellis

Fernando Dourado

Miguel Gama

Lucília Domingues

Biotechnol Biofuels

Enhanced bacterial cellulose production in Komagataeibacter sucrofermentans: impact of different PQQ-dependent dehydrogenase knockouts and ethanol supplementation

增强Komagataeibacter sucrofermentans细菌纤维素生产：不同PQQ依赖性脱氢酶敲除和乙醇补充的影响

Currently, efficient technologies producing useful chemicals from alternative carbon resources, such as methanol, to replace petroleum are in demand. The methanol-utilizing yeast, Komagataella phaffii, is a promising microorganism to produce chemicals from methanol using environment-friendly microbial processes. In this study, to achieve efficient D-lactic acid production from methanol, we investigated a combination of D-lactate dehydrogenase (D-LDH) genes and promoters in K. phaffii. The yeast strain was constructed by integrating a gene cassette containing the identified gene and promoter into the rDNA locus of K. phaffii, followed by post-transformational gene amplification. Subsequently, D-lactic acid production from methanol was evaluated. Among the five D-LDH genes and eight promoters tested, the combination of LlDLDH derived from Leuconostoc lactis and CAT1 and FLD1 promoters was suitable for expression in K. phaffii. GS115_CFL/Z3/04, the best-engineered strain constructed via integration of LlDLDH linked to CAT1 and FLD1 promoters into the rDNA locus and post-transformational gene amplification, produced 5.18 g/L D-lactic acid from methanol. To the best of our knowledge, the amount of D-lactic acid from methanol produced by this engineered yeast is the highest reported value to date when utilizing methanol as the sole carbon source. This study demonstrated the effectiveness of combining different enzyme genes and promoters using multiple promoters with different induction and repression conditions, integrating the genes into the rDNA locus, and further amplifying the genes after transformation in K. phaffii. Using our established method, other K. phaffii strains can be engineered to produce various useful chemicals in the future.

Yoshifumi Inoue

Ryosuke Yamada

Takuya Matsumoto

Hiroyasu Ogino

Enhancing D-lactic acid production by optimizing the expression of D-LDH gene in methylotrophic yeast Komagataella phaffii

通过优化甲基营养酵母近平滑假丝酵母中D-LDH基因表达提高D-乳酸产量的研究

Inspired by the natural symbiotic relationships between diverse microbial members, researchers recently focused on modifying microbial chassis to create artificial coculture systems using synthetic biology tools. An increasing number of scientists are now exploring these systems as innovative biosynthetic platforms for biomass conversion. While significant advancements have been achieved, challenges remain in maintaining the stability and productivity of these systems. Sustaining an optimal population ratio over a long time period and balancing anabolism and catabolism during cultivation have proven difficult. Key issues, such as competitive or antagonistic relationships between microbial members, as well as metabolic imbalances and maladaptation, are critical factors affecting the stability and productivity of artificial coculture systems. In this article, we critically review current strategies and methods for improving the stability and productivity of these systems, with a focus on recent progress in biomass conversion. We also provide insights into future research directions, laying the groundwork for further development of artificial coculture biosynthetic platforms.

Xinyu Song

Yue Ju

Lei Chen

Weiwen Zhang

Strategies and tools to construct stable and efficient artificial coculture systems as biosynthetic platforms for biomass conversion

构建稳定高效的人工共培养系统作为生物质转化生物合成平台的策略与工具

Clostridium thermocellum is a promising candidate for production of cellulosic biofuels, however, its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild-type organism, this reaction uses pyrophosphate (PPi) as an energy cofactor, which is thermodynamically less favorable compared to reactions that use ATP as a cofactor. Previously we showed that replacing the PPi-linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer. In this study, we substituted PPi-pfk with ATP-pfk, deleted the other PPi-requiring glycolytic gene pyruvate:phosphate dikinase (ppdk), and expressed a soluble pyrophosphatase (PPase) and pyruvate kinase (pyk) genes to engineer PPi-free glycolysis in C. thermocellum. We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 to 21.0 g/L) by using PPi-free glycolysis. By engineering PPi-free glycolysis in C. thermocellum, we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in C. thermocellum to increase ethanol titers.

Bishal Dev Sharma

Shuen Hon

Eashant Thusoo

David M. Stevenson

Daniel Amador-Noguez

Adam M. Guss

Lee R. Lynd

Daniel G. Olson

Pyrophosphate-free glycolysis in Clostridium thermocellum increases both thermodynamic driving force and ethanol titers

无焦磷酸的梭菌热纤虫糖酵解过程增加了热力学驱动力和乙醇产量

Abstract Biofilm is a syntrophic community of microorganisms enveloped by extracellular polymeric substances and displays remarkable adaptability to dynamic environments. Implementing biofilm in anaerobic digestion has been widely investigated and applied as it promotes microbial retention time and enhances the efficiency. Previous studies on anaerobic biofilm primarily focused on application in wastewater treatment, while its role has been significantly extended to accelerate the degradation of lignocellulosic biomass, improve gas–liquid mass transfer for biogas upgrading, or enhance resistance to inhibitors or toxic pollutants. This work comprehensively reviewed the current applications of biofilm in anaerobic digestion and focused on impacting factors, optimization strategies, reactor set-up, and microbial communities. Moreover, a full-scale biofilm reactor case from Norway is also reported. This review provides a state of-the- art insight on the role of biofilm in anaerobic digestion. Graphical Abstract

Getachew Birhanu Abera

Erik Trømborg

Linn Solli

Juline M. Walter

Radziah Wahid

Espen Govasmark

Svein Jarle Horn

Nabin Aryal

Lu Feng

Biofilm application for anaerobic digestion: a systematic review and an industrial scale case

生物膜在厌氧消化中的应用：系统综述与工业规模案例

Abstract Background Biorefineries usually focus on the production of low-value commodities, such as bioethanol, platform chemicals or single cell protein. Shifting production to bioactive compounds, such as antimicrobial peptides, could provide an opportunity to increase the economic viability of biorefineries. Results Recombinant production of the antimicrobial peptide pediocin PA-1 in Corynebacterium glutamicum was transferred from yeast extract-based media to minimal media based on lignocellulosic spent sulfite liquor. Induced batch, fed batch, and extended batch process modes were compared for highest pediocin PA-1 production. Conclusion For pediocin PA-1 production on lignocellulosic residues, extended batch cultivation was identified as the optimal process mode, producing up to $$\simeq$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mo>≃</mml:mo> </mml:math> 104 mg/L active pediocin PA-1. Moreover, the production of pediocin PA-1 on this sustainable second generation resource exceeded its state-of-the-art production on yeast extract-based media $$\simeq$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mo>≃</mml:mo> </mml:math> 1.5-fold.

Daniel Waldschitz

Mark-Richard Neudert

Jakob Kitzmüller

Jae Hwi Bong

Yannick Bus

Eva Maria Karner

Peter Sinner

Oliver Spadiut

Antimicrobial peptide production with Corynebacterium glutamicum on lignocellulosic side streams

利用谷氨酸棒杆菌在木质纤维素副产物上生产抗菌肽

Abbreviation	Biotechnol Biofuels
Journal Impact	3.48
Quartiles(Global)	ENERGY & FUELS(Q3)

ISSN	2731-3654
h-index	12

Publication Information	Publisher: BioMed Central Ltd，Publishing cycle: ，Journal Type: journal，Open Access Journals: Yes
Basic data	Year of publication: 2022，Proportion of original research papers: ，Self Citation Rate:， Gold OA Rate: 100.00
Journal Fees	$2890Details
Average review cycle	16 weeks

Biotechnology for Biofuels and Bioproducts

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