Xylose utilization
by Corynebacterium glutamicum is an
essential but unresolved issue in glutamic acid production
from lignocellulose biomass. Coexistence of xylose with inhibitors
requires a selective removal of inhibitors while the xylose is still
well retained in the pretreated lignocellulose feedstock. Not only
is xylose assimilation in C. glutamicum at low efficiency, but also there are unique challenges, which eliminate
the generation of glutamic acid from xylose when lignocellulose is
used. There include excessive biotin content in lignocellulose blocking
intracellular secretion of glutamic acid, complicated organic acid
generation pathways decreasing the glutamic acid conversion yield
from xylose, and transmembrane resistance of xylose limiting the xylose
utilization efficiency. Here, we applied a unique biodetoxification
on pretreated wheat straw solids, which resulted in a complete removal
of inhibitors and a high conservation of xylose sugar. The major focus
of the study is a stepwise metabolic engineering of C. glutamicum to trigger high-titer glutamate production
by coordinated assimilation of xylose and glucose from a typical lignocellulosic
sugar. First, the secretion channel protein MscCG was modified to
initiate glutamic acid secretion in biotin-rich environments from
almost zero glutamic acid accumulation. Next, the byproduct generation
pathways of lactate, acetate, and succinate were knocked out or attenuated
to redirect carbon flux to glutamic acid accumulation. Further overexpression
of the pentose transporter gene araE increased the
xylose utilization rate and glutamic acid production. The finally
obtained C. glutamicum GJ04 produced
39.8 g/L of glutamate from 60.3 g/L of glucose and 38.8 g/L of xylose
in synthetic medium and produced 61.7 g/L of glutamate from 116.1
g/L of glucose and 39.6 g/L of xylose using wheat straw feedstock.
This is the first example of the practical utilization of lignocellulose-derived
xylose and glucose for cellulosic glutamic acid production.
Microbial lipid production from lignocellulose biomass provides an essential option for sustainable and carbon‐neutral supply of future aviation fuels, biodiesel, as well as various food and nutrition products. Oleaginous yeast is the major microbial cell factory but its lipid‐producing performance is far below the requirements of industrial application. Here we show an ultra‐centrifugation fractionation in adaptive evolution (UCF) of Trichosporon cutaneum based on the minor cell density difference. The lightest cells with the maximum intracellular lipid content were isolated by ultra‐centrifugation fractionation in the long‐term adaptive evolution. Significant changes occurred in the cell morphology with a fragile cell wall wrapping and enlarged intracellular space (two orders of magnitude increase in cell size). Complete and coordinate assimilations of all nonglucose sugars derived from lignocellulose were triggered and fluxed into lipid synthesis. Genome mutations and significant transcriptional regulations of the genes responsible for cell structure were identified and experimentally confirmed. The obtained T. cutaneum MP11 cells achieved a high lipid production of wheat straw, approximately five‐fold greater than that of the parental cells. The study provided an effective method for screening the high lipid‐containing oleaginous yeast cells as well as the intracellular products accumulating cells in general.
Lignocellulose is the only feasible carbohydrates feedstock for commercial scale and carbon neutral production of poly(3‐hydroxybutyrate) (PHB) biopolymer by its great abundance and availability. Microbial cell factories for fermentative PHB synthesis are highly restricted by the growth suppression of inhibitors from lignocellulose pretreatment. This study targeted a potential PHB‐producing cell factory Corynebacterium glutamicum owing to its strong inhibitors tolerance. A systematic metabolic engineering was conducted starting with the stable PHB synthesis pathway construction from glucose and xylose, followed by the enhancement of PHB synthesis on PHA synthase activity and stability, cell morphology modification, and growth factors regulation. The relocation of the PHA synthase on the cell membrane guided by secrete signal peptides and cell membrane display motifs increased the PHB content by 2.4 folds. Excessive nitrogen preferentially promoted the PHB synthesis capacity and resulted in the PHB content increased by 13.3 folds. Modification of the genes responsible for cell division changed the cell morphology but the cell size was not enlarged to a PHB accumulation favorable environment. The metabolic engineering of C. glutamicum resulted in a high fermentative production of PHB using wheat straw as feedstock. This study provided an important microbial cell factory choice for PHB production using lignocellulose feedstock.
A preliminary study shows that lysine production from lignocellulose feedstock is feasible, but the conversion of xylose in lignocellulose to lysine remains unsolved. Two technical barriers are responsible for the remaining xylose conversion: one is the xylose loss into the wastewater stream of the biorefinery processing chain, and the other is the lack of efficient lysine-producing strain with xylose utilization. Here, we conducted a new biorefinery approach of consequent dry acid pretreatment and biodetoxification, resulting in zero wastewater generation and then well-preserved xylose. To provide the lysine-producing strain with xylose utilization, we modified the Corynebacterium glutamicum by establishing the xylose assimilation pathway and improving the NADPH cofactor regeneration. The combinational modification of biorefinery processing and strain development led to 31.3 g/L of lysine production with a yield of 0.23 g lysine per gram of wheat straw derived sugars. This study provides a practical method for upgraded lysine production from lignocellulose for future industrial applications.
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