Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

A coculture-coproduction system designed for enhanced carbon conservation through inter-strain CO2 recycling.

Carbon loss in the form of CO2 is an intrinsic and persistent challenge faced during conventional and advanced biofuel production from biomass feedstocks. Current mechanisms for increasing carbon conservation typically require the provision of reduced co-substrates as additional reducing equivalents. This need can be circumvented, however, by exploiting the natural heterogeneity of lignocellulosic sugars mixtures and strategically using specific fractions to drive complementary CO2 emitting vs. CO2 fixing pathways. As a demonstration of concept, a coculture-coproduction system was developed by pairing two catabolically orthogonal Escherichia coli strains; one converting glucose to ethanol (G2E) and the other xylose to succinate (X2S). 13C-labeling studies reveled that G2E + X2S cocultures were capable of recycling 24% of all evolved CO2 and achieved a carbon conservation efficiency of 77%; significantly higher than the 64% achieved when all sugars are instead converted to just ethanol. In addition to CO2 exchange, the latent exchange of pyruvate between strains was discovered, along with significant carbon rearrangement within X2S.

Biomanufacturing of value-added chemicals from lignin.

Lignin valorization faces persistent biomanufacturing challenges due to the heterogeneous and toxic carbon substrates derived from lignin depolymerization. To address the heterogeneous nature of aromatic feedstocks, plant cell wall engineering and 'lignin first' pretreatment methods have recently emerged. Next, to convert the resulting aromatic substrates into value-added chemicals, diverse microbial host systems also continue to be developed. This includes microbes that (1) lack aromatic metabolism, (2) metabolize aromatics but not sugars, and (3) co-metabolize both aromatics and sugars, each system presenting unique pros and cons. Considering the intrinsic complexity of lignin-derived substrate mixtures, emerging and non-model microbes with native metabolism for aromatics appear poised to provide the greatest impacts on lignin valorization via biomanufacturing.

Unravelling the hidden power of esterases for biomanufacturing of short-chain esters.

Microbial production of esters has recently garnered wide attention, but the current production metrics are low. Evidently, the ester precursors (organic acids and alcohols) can be accumulated at higher titers by microbes like Escherichia coli. Hence, we hypothesized that their 'direct esterification' using esterases will be efficient. We engineered esterases from various microorganisms into E. coli, along with overexpression of ethanol and lactate pathway genes. High cell density fermentation exhibited the strains possessing esterase-A (SSL76) and carbohydrate esterase (SSL74) as the potent candidates. Fed-batch fermentation at pH 7 resulted in 80 mg/L of ethyl acetate and 10 mg/L of ethyl lactate accumulation by SSL76. At pH 6, the total ester titer improved by 2.5-fold, with SSL76 producing 225 mg/L of ethyl acetate, and 18.2 mg/L of ethyl lactate, the highest reported titer in E. coli. To our knowledge, this is the first successful demonstration of short-chain ester production by engineering 'esterases' in E. coli.

Corrigendum: Corynebacterium glutamicum as an efficient omnivorous microbial host for the bioconversion of lignocellulosic biomass.

[This corrects the article DOI: 10.3389/fbioe.2022.827386.].

Corynebacterium glutamicum as an Efficient Omnivorous Microbial Host for the Bioconversion of Lignocellulosic Biomass.

Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of ß-ketoadipate pathway in C. glutamicum. 13C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.

Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.

Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli, Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E. coli and B. subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E. coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 µg/ml of protein on day 0 to 464 µg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E. coli transformants expressing mCherry yielded 2.7-fold increase (93 µg/mL to 254 µg/mL) after 10 mins, while 4-fold increase (380 µg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in -80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.